Identification of pyrazinamide-resistant mycobacteria and methods for treating mycobacterial infections

ABSTRACT

Disclosed are methods, probes, primers and kits for identifying pyrazinamide-resistant mycobacteria. These methods can be used to distinguish M. bovis from M. tuberculosis, as well as to identify additional pyrazinamide-resistant mycobacteria. Also disclosed are methods for treating mycobacterial infections by expressing a pncA gene in mycobacteria that infect a mammal, and treating the mammal with pyrazinamide. The invention derives from the discovery of that the molecular basis for pyrazinamide resistance is an alteration in the pncA gene of mycobacteria. The detection of such an alteration is an indicator of PZA-resistant mycobacteria.

FIELD OF THE INVENTION

This invention relates to methods and nucleic acids for identifyingpyrazinamide-resistant mycobacteria, including M. bovis, and fortreating infections with pyrazinamide-resistant mycobacteria.

BACKGROUND OF THE INVENTION

Tuberculosis in humans and other mammals is caused by members of the M.tuberculosis complex, which includes M. tuberculosis, M. bovis, M.africanum, and M. microti. M. bovis and M. tuberculosis are closelyrelated, and share more than 99% sequence identity. Although M. bovis isthe primary cause of tuberculosis in cattle, it is transmissible tohumans, where it can cause tuberculosis.

Conventional methods for treating tuberculosis include administration ofdrugs such as pyrazinamide, isoniazid (INH), rifampicin, streptomycin,and ethambutol. Pyrazinamide (PZA) is particularly useful because itkills certain semi-dormant bacteria that are not affected by otheranti-tuberculosis drugs (McCune et al., 1956, J. Exp. Med. 104: 763-802;Heifets et al., 1992, Am. Rev. Respir. Dis. 145: 1223-1225). Thus,inclusion of PZA with INH and rifampicin considerably shortens thetypical treatment period from 12-18 months to 6 months, and the threedrugs together form the basis of the standard short course chemotherapy(McCune et al., J. Exp. Med. 104, 763-802 (1956); Mitchison, D. S.,Tubercle 66, 219-225 (1985)).

Drug-resistant mycobacteria pose a considerable threat to the control oftuberculosis. Indeed, multidrug-resistant (MDR) strains of M.tuberculosis have caused several fatal outbreaks in both HIV-positiveand HIV-negative individuals (Centers for Disease Control, Florida andNew York, 1988-1991, Mortal, Morbid, Weekly Rep. 40, 585-591 (1991);Bloom, et al., Science 257, 1055-1064 (1992)). Strains of M. bovis arenaturally resistant to PZA, and several strains of M. tuberculosis areknown to have acquired PZA resistance. Examples of acquiredPZA-resistant strains include PZA-R (ATCC 35828) and the clinicalisolate M36470, M3S169, F36946, and Vertullo. Because PZA is a commonlyused anti-tuberculosis drug, the existence of PZA-resistant strains hascomplicated efforts to combat tuberculosis infections. Thus, it isimportant to determine whether the infecting mycobacterium is M. bovisor a PZA-susceptible variant of M. tuberculosis in order to know whetherPZA-based therapy is feasible. Yet, prior to the work described below,the molecular basis of pyrazinamide (PZA) resistance remained unknown.The traditional methods for identifying PZA-resistant mycobacteria, suchas M. bovis, typically involve time-consuming biochemical assays, whichrequire 2-8 weeks to perform.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of the molecular basisfor PZA-resistance in mycobacteria such as M. bovis. PZA resistance isconferred by an alteration(s) in the pncA gene of both naturallyPZA-resistant mycobacteria and acquired PZA-resistant strains ofmycobacteria. The altered pncA genes of PZA-resistant mycobacteria failto encode functional pyrazinamidase polypeptides.

Accordingly, the invention provides methods, primers, probes, and kitsuseful for identifying mycobacteria that are resistant to PZA due toalterations in the pncA gene. Also included within the invention arenucleic acids encoding altered PZase polypeptides that confer PZAresistance and wild-type M. tuberculosis PZase polypeptide. Asubstantially purified wild-type M. tuberculosis PZase polypeptide andsubstantially purified altered PZase polypeptides that confer PZAresistance, and antibodies that specifically bind such polypeptides,also are included in the invention. The identification of PZA-resistantmycobacteria is clinically useful for directing the treatment oftuberculosis patients. In addition, conventional biochemical methods foridentifying PZA-resistant bacteria can produce initially false results.By providing molecular techniques that have a relatively high degree ofaccuracy, the invention facilities the rapid detection of PZA-resistantbacteria, and augments the design of new anti-tuberculosis drugs (e.g.,PZA derivatives), which requires accurate identification of the testedmycobacteria.

Derived from the discovery that M. bovis naturally has an altered pncAgene, the invention provides methods, primers, probes, substantiallypurified polypeptides, and isolated antibodies for distinguishingbetween M. bovis and M. tuberculosis. These methods, nucleic acids,polypeptides, and antibodies are particularly useful as epidemiologicaltools to monitor the spread of bovine tuberculosis to humans ingeographic areas that face tuberculosis outbreaks. For commercialpurposes, the primers and probes of the invention can be packaged into akit.

The invention also provides methods for treating a mammal that isinfected with PZA-resistant mycobacteria. The methods involve (i)introducing into the mycobacteria a functional pncA gene, and (ii)administering PZA to the mammal, thereby killing the mycobacteria.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a listing of the DNA and amino acid sequences of the wild-typepncA gene of M. tuberculosis. The nucleotides that are altered in thealtered pncA genes discussed below are indicated by bold-face type andunderscoring. The complete sequence of the M. tuberculosis pncA gene wasobtained from the 3.2 kb DNA fragment (Erdman strain) using primersdesigned from the sequence of the partial pncA-containing 500 bp PCRproduct.

FIG. 2 is a schematic representation of the pncA gene and the portionsof the gene that are amplified by particular pairs of primers (P1-P8).

FIG. 3A is a restriction map of a 3.2 kb EcoRl-Pstl fragment containingthe pncA. The 3.2 kb fragment was derived from cosmid DNA isolated usinga 500 bp pncA-containing PCR product as a probe. The bold arrowindicates the direction of transcription for the pncA gene. Restrictionendonuclease recognition sites are abbreviated as: E, EcoRi; K, KpnI; P,PstI; S, Smal; and X, XmnI.

FIG. 3B is a pair of nucleotide sequences showing the limited homologybetween the genes encoding M. tuberculosis PZase and E. colinicotinamidase.

FIG. 4 is a pair of photographs showing that the mobility of DNAcorresponding to M. bovis differs from the mobility of DNA correspondingto M. tuberculosis when analyzed by PCR-SSCP. M. bovis: lanes 1-5, 8-15,20-23, 24-31, 36-39, and 42. M. tuberculosis: lanes 6-7, 16-19, 32-35,40-41, and 43-45.

FIG. 5 is a set of photographs showing that the mobility of DNAcorresponding to PZA-resistant M. tuberculosis differs from the mobilityof DNA corresponding to wild-type (PZA-sensitive) M. tuberculosis whenanalyzed by PCR-SSCP. To amplify the DNA electrophoresed in lanes 1-6,primers P3 (SEQ ID NO: 7) and P4 (SEQ ID NO: 8) were used. To amplifythe DNA electrophoresed in lanes 7-9, primers P5 (SEQ ID NO: 9) and P6(SEQ ID NO: 10) were used. Primers P7 (SEQ ID NO: 11) and P8 (SEQ ID NO:12) were used to amplify the DNA that is electrophoresed in lanes 10-14.DNA amplified from PZA-resistant M. tuberculosis strains iselectrophoresed in lanes 1-4, 5, 8, 9, and 12. As a control, DNAamplified from a wild-type (PZA-sensitive) M. tuberculosis strain iselectrophoresed in lanes 5, 7, 10, 11, 13, and 14. The mycobacterialstrains were: M. bovis (ATCC 19210) in lane 1; BCG Tokyo in lane 2(provided by P. Converse, Johns Hopkins University); PZA-resistantclinical isolates of M. tuberculosis from Brazil in lanes 3-6 (providedby S. Morris; FDA); PZA-sensitive M. tuberculosis strain 12646 in lane7; PZA-resistant M. tuberculosis strain derived from type strain H37Rv(ATCC 35828) in lane 8; BCG Tice in lane 9 (provided by P. Converse);PZA-sensitive M. tuberculosis Erdman strain in lanes 10, 11, 13, and 14(H37728, 12646, and M40023); and PZA-resistant M. tuberculosis strainM3169 in lane 12.

A DETAILED DESCRIPTION OF THE INVENTION

Differentiation Between M. bovis and M. tuberculosis: In one aspect,this invention provides a method for differentiating between M.tuberculosis and M. bovis in a sample. The method involves detecting inthe mycobacteria of the sample an altered pncA gene that encodes anaspartic acid residue, rather than a histidine residue, at amino acidposition 57 of the PZase polypeptide encoded by the altered pncA gene.All strains of M. bovis that have been examined to date have thisalteration, whereas this alteration has not been found in any strain ofM. tuberculosis. Thus, an aspartic acid residue at this position isindicative of M. bovis, rather than M. tuberculosis. The aspartic acidresidue at position 57 can be encoded by a change of C to G atnucleotide 169 of the altered pncA gene. Other alterations in nucleotidesequences can also result in an aspartic acid residue at position 57.For example, because of the degeneracy of the genetic code, a change ofC to G at nucleotide 169 combined with a change of C to U at nucleotide171 also will encode an aspartic acid residue at amino acid position 57.

As used herein, the term "altered" refers to any pncA nucleic acid(e.g., a gene) or PZase polypeptide that differs in nucleotide or aminoacid sequence from the wild-type pncA gene of M. tuberculosis (ordegenerate variants thereof) or the wild-type PZase polypeptide of M.tuberculosis. Thus, the M. bovis pncA gene is considered an "altered"pnca gene, even though this gene naturally confers PZA resistance. The"wild-type" M. tuberculosis pncA gene and the wild-type PZasepolypeptide are listed in FIG. 1 (SEQ ID NO: 1 and SEQ ID NO: 2). Whilethe nucleic acid sequences of "degenerate variants" of the pncA genediffer from the illustrated wild-type pncA sequence, the degeneratevariants nonetheless encode a wild-type PZase polypeptide because mostof the 20 natural amino acids are each specified by more than one codon.Thus, a "degenerate variant" of a nucleotide sequence is a nucleotidesequence that encodes the same amino acid sequence as a given nucleotidesequence, but in which at least one codon in the nucleotide sequence isdifferent because two or more different codons can encode the same aminoacid. All degenerate nucleotide sequences are included in the invention,as long as the amino acid sequence of the pncA polypeptide encoded bythe nucleotide sequence is functionally unchanged. In addition, theinvention includes an isolated nucleic acid encoding a polypeptidehaving the biological activity of an amino acid sequence of SEQ ID NO: 2and having at least one epitope for an antibody immunoreactive withPZase polypeptide.

An isolated nucleic acid encoding an M. tuberculosis PZase polypeptideis included within the invention, and useful for production of theprimers and probes described herein. The term "isolated" as used hereinincludes polynucleotides or polypeptides substantially free of othernucleic acids, proteins, lipids, carbohydrates, or other materials withwhich it is naturally associated. The isolated nucleic acids of theinvention encoding M. tuberculosis PZase polypeptide include nucleicacids that encode "conservative variations" of the PZase polypeptide. Aconservative variation as used herein denotes the replacement of anamino acid residue by another, biologically similar, residue. Examplesof conservative variations include the substitution of one hydrophobicresidue, such as isoleucine, valine, leucine, or methionine, foranother, or the substitution of one polar residue for another, such asthe substitution of arginine for lysine, glutamic acid for asparticacid, or glutamine for asparagine, and the like. The term "conservativevariation" also includes the use of a substituted amino acid in place ofan unsubstituted parent amino acid, provided that antibodies raised tothe substituted polypeptide also immunoreact with the unsubstitutedpolypeptide. Also include are isolated nucleic acids that encode aportion or fragment of the PZase polypeptide, as long as the PZasepolypeptide retains a biological activity of the full length PZasepolypeptide, such as the ability to convert PZA to pyrazinoic acid(Konno et al., Am. Rev. Respir. Dis. 95, 461-469 (1967)). Such nucleicacids include naturally-occurring, synthetic, and intentionallymanipulated polynucleotides. For example, portions of the mRNA sequencemay be altered due to alternate promoters for RNA transcription. Asanother example, pncA nucleic acids may be subjected to site-directedmutagenesis. Antisense sequences for pncA also are included.

Any of the various art-known methods for detecting point mutations canbe used to detect the altered M. bovis pncA gene. Now that thealteration in the M. bovis sequence has been identified and appreciated,a person of ordinary skill in molecular biology can readily identify M.bovis using any conventional method for detecting point mutations. Theterm "detecting" as used herein encompasses any means for determiningthe presence of a given gene or polypeptide in a sample. For example, analtered pncA gene can be detected by methods such as, but not limitedto, polymerase chain reaction single-strand conformation polymorphism(PCR-SSCP (Orita et al., 1989, Genomics 5: 874-879)), single-strandconformation polymorphism (SSCP (Orita et al., 1989, Proc. Natl. Acad.Sci. 86: 2766-2770)), DNA sequencing, DNA hybridization (e.g., Southernblotting, dot/slot blotting, colony hybridization), denaturing gradientgel electrophoresis (Myers et al., 1985, Nature, 313: 495-498),ligase-mediated gene detection (Landegren et al., 1988, Science 241:1077-1080), and RNase digestion of an RNA/DNA duplex (Winter et al.,1985, Proc. Natl. Acad. Sci. 82: 7575-7579).

Now that applicants have shown that PZA resistance in M. bovis isconferred by an alteration the pncA gene and PZase, it is recognized forthe first time that immunoassays can be used to distinguish M. bovisfrom M. tuberculosis. Therefore, the invention includes an isolatedpolyclonal or monoclonal antibody that preferentially binds the M. bovisPZase polypeptide. "Isolated antibodies" are those antibodies that areseparated from the animal in which they were raised (e.g., a rabbit ormouse) or antibodies that were produced in vitro. Suitable immunoassaysinclude Western blot analysis, slot or dot blot assays, ELISAs,immunoprecipitation assays, and the like. "Antibody" means animmunoglobulin protein that is capable of binding an antigen. The termantibody is meant to include antibody fragments (e.g., F(Ab')₂, FAb',FAb) capable of binding the epitope or antigen of interest. The term"preferentially binds" means high avidity and/or high affinity bindingof an antibody to a specific antigen or epitope. Antibody binding to anepitope on this specific antigen is stronger than binding of the sameantibody to any other antigen or epitope. In particular, an antibodythat preferentially binds M. bovis PZase binds M. bovis PZase morestrongly than it binds M. tuberculosis PZase. In addition, an antibodythat preferentially binds an antigen or epitope binds that antigen orepitope more strongly than it binds other molecules that may be presentin the same sample as the antigen of interest. Antibodies that bindpreferentially to a polypeptide of interest may be capable of bindingother polypeptides at a weak, yet detectable, level (e.g., 10% or lessof the binding shown to the antigen of interest). Such weak binding, orbackground binding, is readily discernible from the specific antibodybinding to the polypeptide of interest, e.g., by use of appropriatecontrols.

Also included in the invention is a substantially pure M. bovis PZasepolypeptide, or a conservative variant thereof. Such a polypeptide canbe used to produce the above-described antibodies that are useful inimmunoassays. The term "substantially pure polypeptide" means apreparation of a PZase polypeptide that is substantially free from theproteins and other naturally occurring organic molecules with whichPZase is naturally associated. This typically means that the desiredPZase polypeptide constitutes at least 60% of the dry weight of thepreparation. Preferably, the preparation is at least 75%, morepreferably at least 90%, and most preferably at least 99%, by weight,PZase polypeptide. A substantially pure PZase polypeptide may beobtained, for example, by extraction from a natural source (e.g., M.bovis); by expression of a recombinant nucleic acid encoding a PZasepolypeptide; or by chemical synthesis. Purity can be measured and/orobtained by any appropriate method, e.g., column chromatography,polyacrylamide gel electrophoresis, or HPLC.

A protein is substantially free of naturally associated components whenit is separated from those contaminants that accompany it in its naturalstate. Thus, a protein that is chemically synthesized, or produced froma source different from the source from which the protein naturallyoriginates, will be substantially free from its naturally associatedcomponents. Accordingly, substantially pure PZase includes recombinantPZase synthesized, for example, in vitro in a mammalian cell line, in E.coli or another single-celled microorganism, or in insect cells.

PCR-Single-Strand Conformation Polymorphism (PCR-SSCP): The preferredmethod for detecting an alteration in the pncA gene is "PCR-SSCP." Inthis method, a portion of the pncA gene is PCR amplified using primersthat flank the alteration in the gene. The amplified DNA then isanalyzed by gel electrophoresis under non-denaturing conditions. Analteration in the amplified DNA can be detected as an alteration in themobility of the altered DNA through the gel, relative to the mobility ofwild-type DNA. The term "amplifying" means to reproduce a nucleic acid,e.g., by DNA synthesis. For example, an amplified DNA molecule is onethat reproduced such that the total number of copies of the particularDNA molecule is increased. Typically, amplification is accomplished byincubating a nucleic acid polymerase (e.g., DNA polymerase or Taqpolymerase) with non-polymerized nucleotides (e.g., DATP, dCTP, dGTP,and dTTP), and a suitable buffer with a single-stranded nucleic acidtemplate of the DNA to be amplified. For convenience, such amplificationcan be accomplished by "polymerase chain reaction" (PCR), in which anucleic acid is synthesized in the presence of a thermostable polymerase(e.g., Taq polymerase), a large number of suitable nucleic acid primersand non-polymerized nucleotides (see Sambrook et al., supra). In atypical PCR reaction, (1) the strands of a double-stranded DNA moleculeare separated by heating the DNA to produce single-stranded nucleic acidtemplates, (2) the temperature of the reaction is lowered, and thenucleic acid primers are annealed to single-strand nucleic acidtemplates, (3) DNA synthesis ensues, such that a double-strandedmolecule is produced from each original single-stranded template, andsteps (1-3) are repeated for numerous cycles (typically 35 cycles). Nowthat altered pncA genes have been identified and appreciated asindicators of PZA resistance, those of ordinary skill in the art ofmolecular biology can readily use DNA amplification techniques toamplify the pncA gene without undue experimentation.

Typically, the primers for PCR-SSCP are designed such that they canamplify a portion of the gene that is approximately 200 bp in length(e.g., a 100-500 bp, preferably, a 150-200 bp portion). Preferably, thealtered nucleotide is approximately centered within the amplifiedsequence. The exact portion of the pncA gene that is amplified is notcritical to the success of this method, provided that the amplifiedportion encompasses the alteration in the genomic nucleic acid thatconfers PZA resistance (e.g., in the case of M. bovis, the amplifiedportion should include the coding sequence of amino acid 57 of the PZasepolypeptide). An example of a pair of primers that can be used toamplify a suitable portion of the pncA gene for identifying M. bovis is:

5'-GATTGCCGACGTGTCCAGAC-3' (SEQ ID NO: 3) and

5'-ATCAGCGACTACCTGGCCGA-3' (SEQ ID NO: 4).

Sequences that are "substantially complementary" to these sequences alsoare useful for amplifying an appropriate portion of a nucleic acid ofthe mycobacterium that is being identified. This means that the primersmust be sufficiently complementary to hybridize with their respectivestrands under conditions that allow the agent for polymerization toperform. In other words, the primers should have sufficientcomplimentarity with the 5' and 3' sequences flanking a portion of themycobacterial nucleic acid that encompasses an the alteration thatconfers PZA resistance. The mycobacterial sequences to which the primershybridize are considered the "target" flanking 5' and 3' polynucleotidesequences, as they flank the sequence that is to be amplified. Primershaving "substantially the sequence" of the target polynucleotides differin sequence from the target polynucleotide sequence, yet permithybridization of the primer.

PCR amplification of the altered pncA gene can be carried out inaccordance with conventional PCR-SSCP protocols (see Orita et al.,supra). For convenience, a labeled nucleotide (e.g., Sambrook, et al.,Cold Spring Harbor Laboratory Press, 2nd Edition, (1989)) can beincorporated into the DNA that is amplified by PCR. Incorporation of alabeled nucleotide facilitates detection of the amplified DNA directlyin a gel, and obviates the need for transferring the DNA from a gel ontoa membrane. Once the DNA is amplified, it is denatured. Typically,denaturation will be accomplished by heating the DNA to 80°-100° C. for5-10 minutes in the presence of formamide dye (95% formamide, 20 mMEDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol). The heated,amplified DNA then is immediately cooled by incubating the DNA on icefor 5-10 minutes in order to prevent the DNA from renaturing.

In PCR-SSCP, the denatured, amplified DNA is electrophoresed on a gelunder non-denaturing conditions. For amplified DNA fragments ofapproximately 200 bp, an example of a suitable gel is a 20%polyacrylamide/5% glycerol gel (16×20 cm) pre-chilled to 4° C. andelectrophoresed at 4° C. at 5 W in 0.5 X TBE buffer. The electrophoresedDNA then can be detected by conventional methods (e.g., ethidium bromidestaining, DNA hybridization, or in-gel detection of labeled DNA).Typically, the gel will include, as a control, DNA that is amplifiedfrom a known stock of M. tuberculosis and/or M. bovis. A detailedworking example of this method is provided below.

The invention is suitable for distinguishing between M. bovis and M.tuberculosis in any of a variety of samples containing or believed tocontain mycobacteria.

For example, the mycobacteria can be contained within a biological fluidor tissue (e.g., sputum, blood, lung tissue) of a mammal (e.g., a humanor cow). Alternatively, an in vitro culture of mycobacteria, (e.g., atissue culture of mammalian cells that are infected with mycobacteria)can serve as the sample. Because PCR can be used to amplify a portion ofthe altered pncA gene, the sample can, in principle, contain a singlemycobacterium. If desired, mycobacteria obtained from a mammal can becultured in vitro according to conventional methods prior to assayingthe mycobacteria for an altered pncA gene. Likewise, the pncA gene froma mycobacteria sample of interest can be cloned into a genetic vectorprior to assaying for an alteration in the gene, if desired. Inaddition, any of the conventional methods for identifying M. bovis canbe used in conjunction with this method for distinguishing M. bovis fromM. tuberculosis.

Use of Hybridization Methods to Identify M. bovis: As an alternative tousing PCR-SSCP to distinguish M. bovis from M. tuberculosis, traditionalnucleic acid hybridization methods can be used to identify M. bovis.Thus, a nucleic acid probe(s) for use in a nucleic acid hybridizationmethod(s) is included within the invention. Such a "probe" is a nucleicacid molecule (DNA or RNA) that includes a nucleotide sequence that iscomplementary to a portion of (i.e., capable of forming Watson-Crickbase-pairs with part of) a wild-type or altered pncA gene, where theportion includes a nucleotide sequence encoding an aspartic acid residueat amino acid position 57 of the PZase polypeptide. A nucleic acidportion that is substantially complementary to the aforementioned probeis also useful as a probe, and thus is included within the invention. Ina preferred embodiment, the invention includes a probe that iscomplementary to a portion of the M. bovis pncA gene that includes a G,rather than a C, at nucleotide 169. In another preferred embodiment, theprobe is complementary to a portion of the M. tuberculosis pncA genethat includes a C at nucleotide 169. In other words, a probe that iscomplementary to either the wild-type or altered pncA genes is useful,and those skilled in the art will appreciate the results obtained witheither type of probe.

Under typically stringent hybridization conditions, a probe that iscomplementary to the wild-type pncA gene will hybridize to (i.e.,Watson-Crick base-pair with) the pncA gene of M. tuberculosis, but notthe pncA gene of M. bovis. Thus, the inability of such a probe tohybridize to DNA in a sample indicates that the sample includes M.bovis. Inversely, a probe that includes a sequence that is complementaryto the alteration in the M. bovis pncA gene will hybridizepreferentially to the altered M. bovis gene rather than the wild-type M.tuberculosis gene. Those skilled in the art will know how to designappropriate controls for such assays (e.g., include a nucleic acidsample from a known mycobacterium and include a probe that hybridizes toa region of the pncA that is common to M. tuberculosis and M. bovis). Atypical probe consists of a chain of 8 to 20 ribonucleotides ordeoxyribonucleotides, and is complementary to a portion of the pncA genethat is a chain of 8 to 20 deoxyribonucleotides. The position of thealteration along the length of the probe is not critical. If desired,the probes can be detectably labelled. Those of ordinary skill in theart will know or can readily ascertain various techniques for labellingnucleic acid probes.

Identification of PZA-resistant Mycobacteria: The invention alsoprovides methods for identifying PZA-resistant mycobacteria generally.As discussed above, PZA-resistant strains of mycobacteria complicateefforts to treat and contain the spread of tuberculosis. Now that themolecular basis for PZA resistance has been revealed by the experimentsdescribed below, molecular-based assays can be used to identifyPZA-resistant mycobacteria. Included are those mycobacteria that arenaturally resistant to PZA, as well as those mycobacteria that acquirePZA resistance.

In principle, this aspect of the invention is a variation of the abovemethods for distinguishing between M. bovis and M. tuberculosis. For thefirst time, the scientific basis for PZA-resistant strains ofmycobacteria has been discovered by applicants who have shown that thesestrains fail to encode a functional PZase polypeptide due to analteration(s) in the pncA gene. Thus, as above for M. bovis, this aspectof the invention involves detecting an "altered" pncA gene in amycobacterium as an indicator of PZA resistance.

As is described in detail in the examples below, several pncA genealterations that confer PZA resistance have now been identified. Thesealterations are summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                        Mutations in the pncA Gene of PZA-reistant M. tuberculosis                                       PZA suscept-                                                                              MIC For                                        Strain pncA Mutation                                                                             ibility.sup.a                                                                             PZA.sup.b                                                                            MDR.sup.c                               ______________________________________                                        PZA-R  Deletion of -           >500   -                                              G 288.sup.d             μg/ml                                       Vertullo                                                                             Deletion of -           >500   +                                              G 162                   μg/ml                                       F36946 Asp 63 → His                                                                       -           >500   +                                                                      μg/ml                                       M3S169 Gln 141 → Pro                                                                      -           >500   +                                                                      μg/ml                                       M36470 Cys 138 → Ser                                                                      -           >500   +                                                                      μg/ml                                       ______________________________________                                         .sup.a Determined according to the method of McDermott and Tomsett, 1954,     Am. Rev. Tuberc. 70:748-754                                                   .sup.b MIC: Minimal Inhibitory Concentration                                  .sup.c MDR: MultiDrug Resistance against INH and rifampicin                   .sup.d The amino acid positions referred to herein are numbered with          respect to the fulllength wildtype M. tuberculosis PZase polypeptide. The     position of these amino acids may be different in altered PZase               polypeptides.                                                            

In each of strains PZA-R and Vertullo, the deletion of a nucleotideresults in a "-1 frameshift" in the translational reading frame of themRNA encoded to the genes. In other words, because of the -1 frameshiftat position 288, the pncA gene of the PZA-R strain encodesproline-valine-threonine (and so forth), rather thanproline-glycine-aspartic acid at residues 54-56. Similarly, because ofthe -1 frameshift at position 162, the pncA gene of the Vertullo strainencodes lysine-cysteine-leucine (and so forth), rather thanlysine-glycine-alanine at residues 96-98.

Now that alterations in the pncA gene of PZA resistant mycobacteria havebeen identified and shown to be responsible for PZA resistance,PZA-resistant strains of mycobacteria can readily be identified byemploying methods analogous to those described above for identifying M.bovis. In addition, new PZA-resistant strains having as yet unidentifiedspecific alterations the pncA gene can be readily identified by those ofordinary skill without resort to undue experimentation. For example,where a mycobacterium is found to be PZA resistant, but does not containone of the particular alterations in the PZA gene described herein, itis reasonable that such mycobacterium contains a new alteration in itsPZA gene. This alteration can be readily and specifically identified,for example, by nucleic sequencing and comparing the sequence determinedto the wild-type pncA nucleotide sequence.

As is the case for identifying M. bovis, PCR-SSCP is a preferred methodfor identifying PZA-resistant mycobacteria because this method is rapidand reproducible. Included within the invention is a series ofoverlapping primers that is particularly useful for detectingalterations in the pncA gene by PCR-SSCP. These primers permitamplification of the entire pncA gene; thus, they are useful foridentifying any alteration in the pncA gene. These primers include

P1: 5'-GCTGGTCATGTTCGCGATCG-3' (SEQ ID NO: 5);

P2: 5'-TCGGCCAGGTAGTCGCTGAT-3' (SEQ ID NO: 6);

P3: 5'-CGTCGACGTGCAGAACGACT-3' (SEQ ID NO: 7);

P4: 5'-GATTGCCGACGTGTCCAGAC-3' (SEQ ID NO: 8);

P5: 5'-ACCGGACTATTCCTCGTCGT-3' (SEQ ID NO: 9);

P6: 5'-GCGCACACAATGATCGGTGG-3' (SEQ ID NO: 10);

P7: 5'-GCGGCTTCGAAGGAGTCGAC-3' (SEQ ID NO: 1); and

P8: 5'-GCTTTGCGGCGAGCGCTCCA-3' (SEQ ID NO: 12); and sequencessubstantially complementary thereto. The relationship between theseprimers and the pncA gene is shown schematically in FIG. 2; the portionof the pncA gene that is amplified by particular pairs of primers isindicated.

Now that alterations in the pncA gene have been identified andrecognized as conferring PZA resistance to mycobacteria, nucleic acidhybridization methods can also be used to identify PZA-resistantmycobacteria. Accordingly, the invention includes a nucleic acidprobe(s) (DNA or RNA) for identifying a PZA-resistant mycobacterium. Theprobe (typically 8-20 nucleotides) includes a nucleic acid that iscomplementary to a portion (typically 8-20 nucleotides) of an alteredpncA gene of a PZA-resistant mycobacterium, where the portion confersPZA-resistance. Included are nucleic acid probes that are complementaryto a portion of the pncA gene that (i) results in a -1 frameshift atamino acid position 96; (ii) results in a -1 frameshift at amino acidposition 126; (iii) encodes a histidine residue at amino acid position63; (iv) encodes a serine residue at amino acid position 138; and (v)encodes a proline residue at amino acid position 141. Specific examplesinclude those probes that are complementary to a portion of an alteredpncA gene that comprises (i) a deletion of nucleotide 288; (ii) adeletion of nucleotide 162; (iii) a change of G to C at nucleotide 187;(iv) a change of T to A at nucleotide 412; and (v) a change of A to C atnucleotide 422. These probes, when used in hybridization methods, willhybridize preferentially to altered pncA genes. Thus, these probes canbe used to distinguish PZA-resistant mycobacteria from PZA-sensitivemycobacteria by hybridizing the probes to nucleic acids (i.e., DNA orRNA) of the mycobacteria under stringent hybridization conditions.Probes that are complementary to the wild-type pncA sequences can alsobe used to detect PZA-resistant mutants when the probes are designed toinclude a sequence that confers PZA resistance when the sequence isaltered.

The deletion of a single nucleotide in each of the PZA-R and Vertullostrains of M. tuberculosis, described above, results in -1 frameshiftduring synthesis of the altered PZase polypeptide encoded by the alteredpncA gene. Based upon the nucleotide sequence of the pncA gene of thePZA-R strain, the -1 frameshift results in a PZase polypeptide that is126 amino acids in length, rather than a full-length 186 amino acids.Similarly, the -1 frameshift of Vertullo results in a truncated PZasepolypeptide of 117 amino acids. These PZA-resistant strains ofmycobacteria and their homologs can also be detected in immunoassaysthat employ antibodies, especially monoclonal antibodies, thatspecifically bind the carboxy-terminal portion of wild-type PZase thatis missing in the truncated PZase polypeptides. Thus, the altered PZasepolypeptides of PZA-resistant mycobacteria having the alterations failto be bound by such antibodies, even though the altered PZasepolypeptides would be bound by antibodies that specifically bindepitopes at the amino-terminal portion of the polypeptides.

In principle, immunoassays can also be easily developed and used toidentify PZA-resistant strains of mycobacteria, because a single aminoacid alteration also can produce an epitope(s) that is specific to thealtered PZase polypeptide. Also, a truncated, or even elongated, PZasepolypeptide that arises from a frameshift can have an amino acidsequence that, at least in part, differs significantly from that ofwild-type M. tuberculosis PZase polypeptide. Such is the case for thePZase polypeptides of the Vertullo and PZA-R strains of M. tuberculosisbecause of the -1 frameshifts resulting from the deletion of nucleotides162 and 288, respectively. Thus, these PZase polypeptides can bedistinguished from wild-type PZA in immunoassays that employ antibodiesthat preferentially bind the portion of the polypeptide that is alteredas a result of the translational frameshift.

Accordingly, the invention includes isolated antibodies useful inperforming immunoassays for identifying a PZA-resistant mycobacterium.In particular, the invention includes an isolated polyclonal ormonoclonal antibody that preferentially binds an altered PZasepolypeptide that confers PZA resistance, such as a PZase polypeptide ofan M. tuberculosis strain homologous to an M. tuberculosis strainselected from the group consisting of M36470, M3S169, F36946, Vertullo,and PZA-R. The term "homologous" is meant to include strains ofmycobacteria that have PZase polypeptides that are identical to, orconservative variations of, the PZase polypeptides of the recitedstrains. Such homologous strains of mycobacteria can differ from therecited strains at any genetic locus, provided that the homologousstrain is capable of causing respiratory tuberculosis in mammals, and isrendered PZA-resistant by an altered pncA gene. Included by the term"homologous" are the recited strains themselves, namely, M36470, M3S169,F36946, Vertullo, and PZA-R.

The invention also includes a substantially pure altered PZasepolypeptide that confers PZA resistance, or a conservative variantthereof. Specific examples of such polypeptides are the PZasepolypeptides of the M36470, M3S169, F36946, Vertullo, and PZA-R strainsof M. tuberculosis and strains homologous thereto. In other words, anyPZase polypeptides having the alterations summarized in Table 1 areexpected to confer PZA resistance, and are included within theinvention.

In another aspect, the invention provides a method for treating a mammalthat is infected with PZA-resistant mycobacteria (e.g., M. bovis orPZA-resistant M. tuberculosis); thus, this aspect of the inventionprovides a method for treating tuberculosis. The method involvesintroducing a pncA gene that encodes a functional PZase polypeptide intoat least a portion (preferably at least 10%, more preferably at least50%, and most preferably at least 75%) of the PZA-resistant mycobacteriathat infect the mammal. A PZase polypeptide is considered "functional"if it retains a biological activity of the full length PZasepolypeptide, namely the ability to convert PZA to pyrazinoic acid.Expression of the pncA gene in the PZA-resistant mycobacteria rendersthe mycobacteria sensitive to PZA. The mammal then is administered atherapeutically effective amount of PZA, which inhibits or kills atleast a portion of the mycobacteria that infect the mammal. A treatmentthat "inhibits" the mycobacteria slows or prevents the growth orreproduction of the bacteria inside the infected mammal and/or decreasesor prevents the production of release of mycobacterial toxins into thecells of the infected mammal. Generally, the terms "treating,""treatment," and the like are used herein to mean obtaining a desiredpharmacologic and/or physiologic effect. The effect may be prophylacticin terms of completely or partially preventing a mycobacterial infectionor disease (e.g., tuberculosis) or sign or symptom thereof, and/or maybe therapeutic in terms of a partial or complete cure for an infectionor disease and/or adverse effect attributable to the infection ordisease. "Treating" as used herein covers any treatment of an infectionor disease in a mammal, particularly a human, and includes:

(a) preventing the disease from occurring in a subject that may bepredisposed to the disease, but has not yet been diagnosed as having it;

(b) inhibiting the infection or disease, i.e., arresting itsdevelopment; or

(c) relieving or ameliorating the infection or disease, i.e., causeregression of the infection or disease. The invention is thus directedto treating patients who are afflicted with a mycobacterial infection ortuberculosis. More specifically, "treatment" is intended to meanproviding a therapeutically detectable and beneficial effect on apatient suffering from a mycobacterial infection or tuberculosis.

The term "introducing" a pncA gene into a mycobacterium means to inserta pncA gene into the mycobacterium such that the pncA gene is availableto be expressed by the cellular machinery (e.g., polymerases andribosomes) of the mycobacterium. The pncA gene can be introduced intomycobacteria by using standard recombinant DNA techniques to insert thepncA gene into a mycobacteriophage, such as mycobacteriophage L5, L1, orD29 (Hatfull and Jacobs, Mycobacteriophages: Cornerstones ofMycobacterial Research. In Tuberculosis. (ed) B. R. Bloom, ASM Press,Washington, D.C.). A therapeutically effective amount of the pncA genethen is administered to the infected mammal, and a therapeuticallyeffective amount of PZA is administered to the mammal. The term"therapeutically effective" means sufficient pncA gene to produce PZaseto thereby render the mycobacteria resistant to PZA. It is believed thatone copy of the pncA gene per organism would render the organismsensitive to PZA. Various means can be used to "administer" PZA or apncA gene to a mammal as a therapeutic. Generally, the therapeutic to bedelivered can be formulated into a pharmaceutical composition byadmixture with a pharmaceutically acceptable non-toxic excipient orcarrier (e.g., saline). In practicing the invention, the therapeutic canbe prepared for use in parenteral administration (e.g., for intravenousinjection or intra-arterial injection). Preferably, the pncA gene isadministered to the mammal intranasally or intrabronchially, e.g., as aliquid or aerosol formulation (e.g., nasal drops or spray) in apharmaceutically acceptable excipient. Preferably, PZA is administeredorally or according to other conventional methods. While the optimaltherapy regime can be determined for each treated mammal (e.g., a humanor cow), a typical regime involves administration of 10⁶ to 10⁸ pfc ofmycobacteriophage carrying the pncA gene, 1 to 7 times weekly for aperiod of 3 to 12 (preferably 6-12) months, or until the signs andsymptoms of the mycobacterial infection or tuberculosis are ameliorated.PZA typically is administered daily (in combination with isoniazid andrifampicin, if desired) for the first two months although alternativePZA treatment regimes can be used (Mitchison et al., 1985, Tubercle 66:219-225). If desired, this therapy regime can be used in conjunctionwith conventional methods, such as administration of at least one othermycobacterial therapeutic agent (e.g., isoniazid and/or rifampicin).

Included within the invention is a kit(s) for distinguishing M. bovisfrom M. tuberculosis or for identifying PZA-resistant mycobacteria. Sucha kit is a package that carries a container(s) that contains any or allof the primers and probes described above. Preferably, the kit alsocontains isolated nucleic acid(s) corresponding to all or a portion ofthe wild-type M. tuberculosis pncA gene and/or an altered pncA gene(e.g., from M. bovis or one of the acquired PZA-resistant M.tuberculosis strains, such as Vertullo). A kit can contain theabove-described monoclonal or polyclonal antibodies useful foridentifying PZA-resistant mycobacteria. Typically, the nucleic acids,antibodies, and polypeptides that are contained within the kits arepresent in a measured amount that is indicated on a label(s) on thecontainer within the kit. In addition, a typical kit includes a set ofinstructions for practicing the methods of the invention. Nucleic acids,antibodies, and polypeptides that are useful as controls can also beincluded in a kit of the invention.

SUMMARY OF THE EXAMPLES

Several working examples are provided below. These examples describe (i)the cloning of the M. tuberculosis pncA gene, (ii) identification of thealterations in the pncA gene that confer PZA resistance, (iii) use ofthe pncA gene to render PZA-resistant M. tuberculosis sensitive to PZA,(iv) use of PCR-SSCP to rapidly differentiate between M. bovis and M.tuberculosis, and (v) use of PCR-SSCP to identify PZA-resistant M.tuberculosis strains. Before describing the examples in further detail,a description of the materials and methods used in these examples isprovided.

Materials and Methods

Mycobacterial strains and DNA. Strains of M. tuberculosis, M. bovis, andBCG were grown in 7H9 liquid medium with ADC (Albumin-Dextrose-Catalase)enrichment at 37° C. for 2-4 weeks. The PZA-resistant M. tuberculosisstrain (ATCC 35828) derived from type strain H37Rv was obtained fromAmerican Type Culture Collection. PZA-resistant clinical isolates wereprovided by L. Heifets (National Jewish Center for Immunology andRespiratory Diseases, Denver, Colo.), J. Belisle and P. Brennan(Colorado State University). Genomic DNA from M. tuberculosis strainsand other mycobacterial species was isolated as described previously(Zhang, et al., Infect. Immun. 60, 2160-2165 (1992)). Genomic DNA fromM. bovis strains was provided by V. P. Shankar (Texas A & M UniversityHealth Science Center).

Cloning and characterization of the M. tuberculosis pncA gene. To clonethe M. tuberculosis pncA gene, degenerate primers were designed based onthe putative E. coli nicotinamidase amino acid sequence (Jerlstrom, etal., Gene 78, 37-46 (1989)). When cloned into a plasmid vector, theputative E. coli pncA indeed expresses functional nicotinamidaseactivity upon transformation into a Salmonella typhimurium pncA mutantstrain JF49 (Foster et al., J. Bacteriol., 137, 1165-1175 (1979)). Theforward primer was designed from the 9-17 amino-acid residues VDLQNDFCA(SEQ ID NO: 13), and the reverse primer from the 168-179 amino-acidresidues GYKVNVITDGC (SEQ ID NO: 14), of the E. coli enzyme (Jerlstrom,et al., Gene 78, 37-46 (1989)). PCR was performed using the aboveprimers with genomic DNA from a PZA-susceptible M. tuberculosis strainas template (Saiki, et al., Science 239, 487-491 (1988)). A 500 bp PCRproduct was obtained, and sequence analysis showed that it contained apartial open reading frame with homology to the E. coli nicotinamidasegene described above. A cosmid clone containing the complete M.tuberculosis pncA gene was isolated using the ³² P-labelled 500 bp PCRproduct containing the partial pncA gene as a probe, by screening anintegrating mycobacterial shuttle cosmid DNA library constructed fromthe M. tuberculosis Erdman strain (provided by W. R. Jacobs, AlbertEinstein College of Medicine, New York). Restriction mapping of thecosmid DNA, followed by Southern blot analysis using the 500 bp PCRproduct as a probe, and transformation studies localized the M.tuberculosis pncA gene on a 3.2 kb EcoRl-PstI fragment, whichsubsequently was subcloned into pUC19. The standard molecular biologytechniques were carried out as described previously (Sambrook, et al.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

The complete M. tuberculosis pncA sequence (from M. tuberculosis Erdmanstrain) (accession number under application) was determined from the 3.2kb EcoRI-PstI fragment using primers derived from sequences of the 500bp pncA-containing PCR product. The determination of pncA sequences fromM. bovis, BCG and other M. tuberculosis strains was performed by PCRdirect sequencing using primers designed from the above-describedcomplete M. tuberculosis pncA sequence in an automatic DNA sequencer.The sequence homology alignment between M. tuberculosis and E. coli PncAsequences was performed using a FASTA algorithm.

Transformation of tubercle bacilli. The pncA plasmid construct fortransformation of PZA-resistant BCG and H37Rv was made as follows. The3.2 kb EcoRI-PstI fragment containing the functional M. tuberculosispncA was cloned into the hygromycin mycobacterial shuttle vector p16R1(Garbe, et al., Microbiology 140, 133-138 (1994)) as described(Sambrook, et al., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989). Both the p16R1 3.2 kb pncA construct and the cosmidDNA containing the functional M. tuberculosis pncA gene, along with thevector controls, were transformed by electroporation into the naturallyPZA-resistant M. bovis BCG and PZA-resistant M. tuberculosis H37Rv asdescribed (Zhang, et al., Mol. Microbiol. 88, 521-524 (1993)).

PZase enzyme assay. Pyrazinamidase activity was assayed according to themethod of Wayne (Wayne, Am. Rev. Respir. Dis. 109, 147-151 (1974)).Briefly, a heavy bacterial inoculum (several loopfuls) was inoculatedonto the surface of Dubos agar (Difco) containing 0.1 mg/ml PZA in atest tube, which was then incubated at 37° C. for 4 days. Twomilliliters of 1 % ferrous ammonium sulfate were added, and the testtubes were incubated at 4° C. for 1-2 hours. A positive PZase activityappeared as a brownish band in the agar surface. A positive culture(PZA-sensitive M. tuberculosis strain H37Rv) and a negative culture (BCGPasteur) were included as controls.

PZA susceptibility testing. The susceptibility to PZA of recombinantmycobacterial strains was tested in 7H9 liquid medium adjusted to acidpH (5.6) as described elsewhere (McDermott, et al., Am. Rev. Tuberc. 70,748-754 (1954)). Briefly, 2 week old liquid mycobacterial cultures(about 10⁷⁻⁸ bacilli) were diluted at 10⁻³ and 10⁻⁵ in acid 7H9 liquidmedium (pH 5.6) containing 50, 100, 200, 500, 1000 μg/ml PZA inEppendorf tubes. Each strain tested for PZA susceptibility was induplicate. The Eppendorf tubes were then incubated at 37° C. for 2-3weeks when the extent of growth inhibition was assessed. PZA susceptibleH37Ra or H37Rv, and PZA-resistant BCG were included as sensitive andresistant controls, respectively.

Macrophage infections with mycobacteria and assay of PZA susceptibilityof intracellular mycobacteria were performed as described (Skinner, etal., Antimicrob Agent Chemother, 38, 2557-2563 (1994)). Briefly, 5×10⁵J774 murine macrophages were infected with 2-5×10⁶ bacteria in 24-welltissue culture plates. Each mycobacterial strain used for macrophageinfection was in triplicate. After infection at 37° C. for 1-2 hours,the extracellular bacteria were washed off with PBS twice. Tissueculture medium (RPMI 1640) containing various concentrations of PZA orstreptomycin was added to the infected macrophages and the plates wereincubated in a CO₂ incubator at 37° C. for 1-2 weeks. The number ofintracellular bacteria was counted under the microscope and expressed asan average of at least 50 infected macrophages per well for all threetriplicate wells for each bacterial strain.

EXAMPLES

Cloning and characterization of the M. tuberculosis pncA gene. The M.tuberculosis pncA gene was cloned by PCR using degenerate primers basedon the amino acid sequence derived from the E. coli nicotinamidase gene(pncA) (Jerlstrom, et al., Gene 78, 37-46 (1989)). The M. tuberculosispncA was initially cloned on a 500 bp PCR product from a PZA-susceptibleM. tuberculosis strain. Sequence analysis showed that the PCR productcontained a partial open reading frame with homology to the E. colinicotinamidase sequence (Jerlstrom, et al., Gene 78, 37-46 (1989)). Toobtain the complete M. tuberculosis pncA gene, an M. tuberculosisintegrating cosmid DNA library was screened using the 500 bppncA-containing PCR product as a probe. A pncA-hybridizing cosmid clonewas isolated and used to transform BCG, an attenuated vaccine strainderived from M. bovis, in order to confirm the identity of the putativeM. tuberculosis pncA gene. Indeed, the cosmid DNA containing theputative M. tuberculosis pncA gene conferred PZase activity to BCG, anatural mutant defective in PZase. The integration of the pncA cosmidDNA into M. bovis BCG genome was confirmed by Southern analysis. Thefunctional M. tuberculosis pncA gene was localized on a 3.2 kbEcoRI-PstI fragment (FIG. 3A), by restriction mapping of the cosmid DNAin combination with BCG transformation studies using DNA constructsderived from the cosmid DNA insert. To confirm that the PZase activityis due to the pncA gene, but not to other DNA in the pncA upstreamregion on the 3.2 kb EcoRI-PstI fragment, BCG was transformed with the2.3 kb EcoRI-Smal DNA construct that contains the pncA upstream regionand partial pncA gene; however, no PZase activity was detectable in theBCG transformant. Sequence analysis of the 2.3 kb EcoRI-SmaI fragmentdoes not show significant open reading frames with homology to otherknown proteins in the database. Transformation of BCG with a constructthat contains the pncA gene alone with its 120 bp upstream sequence as aPCR fragment gave functional expression of PZase activity, indicatingthat the PZase activity was indeed conferred by the pncA gene.

Sequence analysis revealed that the M. tuberculosis pncA gene (558 bp)encoded a protein of 186 amino acids (FIG. 1), with 35.5% overall aminoacid identity to the E. coli nicotinamidase (Jerlstrom, et al., Gene 78,37-46 (1989)) (FIG. 3B). The predicted molecular mass of the M.tuberculosis PZase is approximately 20 kilodaltons (kD), and is smallerthan the E. coli homolog which consists of 213 amino acids with a sizeof 23 kD (Jerlstrom, et al., Gene 78, 37-46 (1989)).

Identification of mutations in the pncA gene of PZA-resistant strains.Using the 3.2 kb EcoRI-PstI pncA containing DNA fragment as a probe,Southern blotting analysis was performed on a panel of 8 PZA-resistantM. tuberculosis strains, 3 M. bovis strains and 3 BCG substrains. Noneof these strains had any gross deletions of the pncA gene or restrictionfragment length polymorphism. M. bovis, as a species, lacks PZase(Konno, et al., Nature, 184, 1743-1744 (1959)) (Konno, et al., Am Rev.Respir. Dis. 95, 461-469 (1967), yet it nonetheless had the pncA gene,as evidenced by the same sized hybridization fragment as that in M.tuberculosis. The M. tuberculosis pncA gene did not hybridize withgenomic DNA from mycobacterial species that do not cause respiratorytuberculosis, such as M. smegmatis, M. vaccae and M. kansasii.

The sequence of the pncA gene from naturally PZA-resistant M. bovis andan a panel of acquired PZA-resistant M. tuberculosis strains wasdetermined. Surprisingly, three M. bovis strains (Ravenel, and twoveterinary isolates) and three BCG substrains (Pasteur, Copenhagen,Glaxo) all had the same single point mutation in the pncA gene, changingfrom "C" to "G" at nucleotide position 169, which caused a substitutionof Histidine (CAC) for Aspartic Acid (GAC) at amino acid position 57 ofthe PZase polypeptide (FIG. 1). Because no other mutations were found inthe pncA gene, this particular substitution is concluded to have causedthe defective PZase in these M. bovis and BCG strains. Consistent withthis conclusion is the observation that, the BCG strains transformedwith the 3.2 kb EcoRI-PstI construct containing the functional M.tuberculosis pncA gene restored PZase sensitivity.

Of the 8 sequenced M. tuberculosis strains that are PZA-resistant inconventional tests, 5 strains had point mutations within the pncA gene(see Table 1 above). The H37Rv, CSU20, and CSU25 strains lackedmutations in the pncA gene. The PZA-resistant strain PZA-R (ATCC 35828,PZase-negative) derived from H37Rv, and one MDR strain "Vertullo" eachhad a single nucleotide "G" missing at nucleotide positions 288 and 162,respectively, leading to premature termination that caused truncatedpolypeptides with no PZase activity. Three other PZA-resistant clinicalisolates of M. tuberculosis (MDR strains) contained missense mutationsin the pncA gene, causing substitutions at the following amino acidpositions: Asp63 (GAC)→His (CAC), Cysl38 (TGT)→Ser (AGT), Glnl41(CAG)→Pro (CCG). These data suggest that these substitutions areresponsible for PZA-resistance and the defective PZase enzyme activityin these PZA-resistant strains. Of the 3 apparently PZA-resistantstrains (CSU20, CSU25, and H7728) that did not have mutations in thepncA gene, re-testing of the minimal inhibitory concentration (MIC) ofPZA indicated that these three strains are "false resistant," and are infact susceptible to PZA with positive PZase activity.

Use of PCR-SSCP to distinguish M. bovis from M. tuberculosis. In thisexample, PCR was performed using the following cycling parameters: 95°C. for 5 minutes, followed by 30 cycles of (95° C. for 1 minute, 55° C.for 1 minute, and 72° C. for 1 minute). The PCR reaction included 1 XPCR Buffer (GIBCO BRL), dNTPs at 100μ mole, primers at 0.1 μg,mycobacterial genomic DNA 0.1-0.5 μg (V. P. Shankar; Texas A & MUniversity Health Science Center), and 2.5 units of Taq DNA polymerase(GIBCO BRL). The primers used for detection of M. bovis were:

5'-GATTGCCGACGTGTCCAGAC-3' (SEQ ID NO: 3) and

5'-ATCAGCGACTACCTGGCCGA-3' (SEQ ID NO: 4), corresponding to nucleotides91-110 and 270-251, respectively, of the pncA gene. When used together,these primers amplify a 180 bp DNA fragment.

Polyacrylamide gel electrophoresis was used to show the single strandconformation polymorphism in the amplified DNA. The PCR products (10 μl,containing 0.5-1.0 μg DNA) were denatured by boiling for 5-10 minutes informamide dye. The boiled DNA was immediately incubated on ice for 5-10minutes. The denatured DNA then was electrophoresed on a 20%polyacrylamide/5% glycerol gel (16×20 cm) that was pre-cooled to 4° C.The gel was electrophoresed in 0.5 X TBE buffer at a constant power of 5W at 4° C. overnight. The gel then was stained with ethidium bromide(0.5 μg/ml), and the SSCP bands were visualized under UV light. As isillustrated in FIG. 4, the mobility of DNA corresponding to M. bovisdiffers from the mobility of DNA corresponding to M. tuberculosis. Thus,M. bovis strains can be rapidly differentiated from M. tuberculosis byPCR-SSCP, based on the detection of a characteristic M. bovis alterationin the pncA gene.

Use of PCR-SSCP to Identify PZA-resistant Strains of M. tuberculosis. Inthis example, PCR-SSCP was performed essentially as described above,except that the primers and mycobacterial DNA differed. The primers usedwere:

P3: 5'-CGTCGACGTGCAGAACGACT-3' (SEQ ID NO: 7);

P4: 5'-GATTGCCGACGTGTCCAGAC-3' (SEQ ID NO: 8);

P5: 5'-ACCGGACTATTCCTCGTCGT-3' (SEQ ID NO: 9);

P6: 5'-GCGCACACAATGATCGGTGG-3' (SEQ ID NO: 10);

P7: 5'-GCGGCTTCGAAGGAGTCGAC-3' (SEQ ID NO: 11); and

P8: 5'-GCTTTGCGGCGAGCGCTCCA-3' (SEQ ID NO: 12).

By using these primers in pairs (P3 with P4; P5 with P6; and P7 withP8), overlapping portions (˜200 bp) of the pncA gene are amplified,which together correspond to nearly the entire pncA gene (FIG. 2). Byusing a set of primers that amplify several portions of the pncA, onecan use PCR-SSCP to identify a PZA-resistant mycobacterium, even if onedoes not know precisely where in the pncA gene an alteration is located.

As was the case for M. bovis above, polyacrylamide gel electrophoresiswas used to show the single strand conformation polymorphism in theamplified DNA after the DNA was denatured and cooled on ice. As isillustrated in FIG. 5, the mobility of DNA corresponding toPZA-resistant M. tuberculosis differs from the mobility of DNAcorresponding to wild-type (PZA-sensitive) M. tuberculosis. Thus,PZA-resistant strains of M. tuberculosis can be rapidly identified byPCR-SSCP.

Transformation of PZA-resistant strains with a functional pncA gene. Asis shown below, introduction of a functional pncA gene into aPZA-resistant M. bovis or M. tuberculosis renders the mycobacteriumPZA-sensitive. In this case, the susceptibility of M. bovis BCG and theM. tuberculosis strain PZA-R were tested. The M. bovis BCG is anaturally PZA-resistant strain, and the M. tuberculosis strain PZA-R isan acquired resistance strain derived from H37Rv. These twoPZA-resistant strains were transformed with the 3.2 kb EcoRI-PstIplasmid construct that contains the functional pncA gene. In both cases,the resulting pncA transformants expressed PZase enzyme activity andbecame susceptible to PZA (MIC=50 μg ml⁻¹) at acid pH (5.5) in vitro. Incontrast, BCG and PZA-R vector control strains remained PZA-resistant(MIC>1000 μg ml⁻) and PZase-negative.

The pncA gene can also be used to render intracellular M. tuberculosissusceptible to PZA. In this case, the recombinant M. bovis was testedfor PZA-susceptibility while inside macrophages. Indeed, the recombinantBCG expressing the M. tuberculosis PZase became susceptible to PZA inJ774 macrophages. By contrast, the control BCG remained resistant to PZAat various drug concentrations (Table 2). At high PZA concentrations(500 μg ml⁻¹), the control BCG was also inhibited slightly, but not asmuch as the recombinant BCG. In a parallel control experiment withstreptomycin, both the recombinant BCG and the vector control BCG wereequally inhibited by streptomycin in J774 macrophages (Table 2),indicating that the PZA susceptibility of the recombinant BCG is due toexpression of the PZase activity conferred by the M. tuberculosis pncAgene.

                  TABLE 2                                                         ______________________________________                                        PZA Susceptibility of Recombinant BCG in J774 Macrophage                                           Number of bacilli                                                 Drug        per macrophage                                                    concentration                                                                             BCG recombinant                                                                            BCG vector                                  Drugs    (μg/ml)  (PZase.sup.+)                                                                              (PZase.sup.-)                               ______________________________________                                        PZA      0           >20          >20                                                  25          7            >20                                                  200         5            15                                                   500         2            15                                          Streptomycin                                                                           0           >20          >20                                                  25          8            9                                                    50          4            7                                                    100         4            4                                           ______________________________________                                    

Although the invention has been described with reference to thepresently preferred embodiment, it should be understood that variousmodifications can be made without departing from the spirit of theinvention. Accordingly, the invention is limited only by the followingclaims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 16                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 561 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..561                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATGCGGGCGTTGATCATCGTCGACGTGCAGAACGACTTCTGCGAGGGT48                            MetArgAlaLeuIleIleValAspValGlnAsnAspPheCysGluGly                              151015                                                                        GGCTCGCTGGCGGTAACCGGCGGCGCCGCGCTGGCCCGCGCCATCAGC96                            GlySerLeuAlaValThrGlyGlyAlaAlaLeuAlaArgAlaIleSer                              202530                                                                        GACTACCTGGCCGAAGCGGCGGACTACCATCACGTCGTGGCAACCAAG144                           AspTyrLeuAlaGluAlaAlaAspTyrHisHisValValAlaThrLys                              354045                                                                        GACTTCCACATCGACCCGGGTGACCACTTCTCCGGCACACCGGACTAT192                           AspPheHisIleAspProGlyAspHisPheSerGlyThrProAspTyr                              505560                                                                        TCCTCGTCGTGGCCACCGCATTGCGTCAGCGGTACTCCCGGCGCGGAC240                           SerSerSerTrpProProHisCysValSerGlyThrProGlyAlaAsp                              65707580                                                                      TTCCATCCCAGTCTGGACACGTCGGCAATCGAGGCGGTGTTCTACAAG288                           PheHisProSerLeuAspThrSerAlaIleGluAlaValPheTyrLys                              859095                                                                        GGTGCCTACACCGGAGCGTACAGCGGCTTCGAAGGAGTCGACGAGAAC336                           GlyAlaTyrThrGlyAlaTyrSerGlyPheGluGlyValAspGluAsn                              100105110                                                                     GGCACGCCACTGCTGAATTGGCTGCGGCAACGCGGCGTCGATGAGGTC384                           GlyThrProLeuLeuAsnTrpLeuArgGlnArgGlyValAspGluVal                              115120125                                                                     GATGTGGTCGGTATTGCCACCGATCATTGTGTGCGCCAGACGGCCGAG432                           AspValValGlyIleAlaThrAspHisCysValArgGlnThrAlaGlu                              130135140                                                                     GACGCGGTACGCAATGGCTTGGCCACCAGGGTGCTGGTGGACCTGACA480                           AspAlaValArgAsnGlyLeuAlaThrArgValLeuValAspLeuThr                              145150155160                                                                  GCGGGTGTGTCGGCCCATACCACCGTCGCCGCGCTGGAGGAGATGCGC528                           AlaGlyValSerAlaHisThrThrValAlaAlaLeuGluGluMetArg                              165170175                                                                     ACCGCCAGCGTCGAGTTGGTTTGCAGCTCCTGA561                                          ThrAlaSerValGluLeuValCysSerSer*                                               180185                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 186 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetArgAlaLeuIleIleValAspValGlnAsnAspPheCysGluGly                              151015                                                                        GlySerLeuAlaValThrGlyGlyAlaAlaLeuAlaArgAlaIleSer                              202530                                                                        AspTyrLeuAlaGluAlaAlaAspTyrHisHisValValAlaThrLys                              354045                                                                        AspPheHisIleAspProGlyAspHisPheSerGlyThrProAspTyr                              505560                                                                        SerSerSerTrpProProHisCysValSerGlyThrProGlyAlaAsp                              65707580                                                                      PheHisProSerLeuAspThrSerAlaIleGluAlaValPheTyrLys                              859095                                                                        GlyAlaTyrThrGlyAlaTyrSerGlyPheGluGlyValAspGluAsn                              100105110                                                                     GlyThrProLeuLeuAsnTrpLeuArgGlnArgGlyValAspGluVal                              115120125                                                                     AspValValGlyIleAlaThrAspHisCysValArgGlnThrAlaGlu                              130135140                                                                     AspAlaValArgAsnGlyLeuAlaThrArgValLeuValAspLeuThr                              145150155160                                                                  AlaGlyValSerAlaHisThrThrValAlaAlaLeuGluGluMetArg                              165170175                                                                     ThrAlaSerValGluLeuValCysSerSer                                                180185                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GATTGCCGACGTGTCCAGAC20                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATCAGCGACTACCTGGCCGA20                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GCTGGTCATGTTCGCGATCG20                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TCGGCCAGGTAGTCGCTGAT20                                                        (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CGTCGACGTGCAGAACGACT20                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GATTGCCGACGTGTCCAGAC20                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       ACCGGACTATTCCTCGTCGT20                                                        (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GCGCACACAATGATCGGTGG20                                                        (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GCGGCTTCGAAGGAGTCGAC20                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GCTTTGCGGCGAGCGCTCCA20                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ValAspLeuGlnAsnAspPheCysAla                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GlyTyrLysValAsnValIleThrAspGlyCys                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 186 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      MetArgAlaLeuIleIleValAspValGlnAsnAspPheCysGluGly                              151015                                                                        GlySerLeuAlaValThrGlyGlyAlaAlaLeuAlaArgAlaIleSer                              202530                                                                        AspTyrLeuAlaGluAlaAlaAspTyrHisHisValValAlaThrLys                              354045                                                                        AspPheHisIleAspProGlyAspHisPheSerGlyThrProAspTyr                              505560                                                                        SerSerSerTrpProProHisCysValSerGlyThrProGlyAlaAsp                              65707580                                                                      PheHisProSerLeuAspThrSerAlaIleGluAlaValPheTyrLys                              859095                                                                        GlyAlaTyrThrGlyAlaTyrSerGlyPheGluGlyValAspGluAsn                              100105110                                                                     GlyThrProLeuLeuAsnTrpLeuArgGlnArgGlyValAspGluVal                              115120125                                                                     AspValValGlyIleAlaThrAspHisCysValArgGlnThrAlaGlu                              130135140                                                                     AspAlaValArgAsnGlyLeuAlaThrArgValLeuValAspLeuThr                              145150155160                                                                  AlaGlyValSerAlaAspIleThrValAlaAlaLeuGluGluMetArg                              165170175                                                                     ThrAlaSerValGluLeuValCysSerSer                                                180185                                                                        (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 213 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: Not Relevant                                                (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      MetProProArgAlaLeuLeuLeuValAspLeuGlnAsnAspPheCys                              151015                                                                        AlaGlyGlyAlaLeuAlaValProGluGlyAspSerThrValAspVal                              202530                                                                        AlaAsnArgLeuIleAspTrpCysGlnSerArgGlyGluAlaValIle                              354045                                                                        AlaSerGlnAspTrpHisProAlaAsnHisGlySerPheAlaSerGln                              505560                                                                        HisGlyValGluProTyrThrProGlyGlnLeuAspGlyLeuProGln                              65707580                                                                      ThrPheTrpProAspHisCysValGlnAsnSerGluGlyAlaGlnLeu                              859095                                                                        HisProLeuLeuHisGlnLysAlaIleAlaAlaValPheHisLysGly                              100105110                                                                     GluAsnProLeuValAspSerTyrSerAlaPhePheAspAsnGlyArg                              115120125                                                                     ArgGlnLysThrSerLeuAspAspTrpLeuArgAspHisGluIleAsp                              130135140                                                                     GluLeuIleValMetGlyLeuAlaThrAspTyrCysValLysPheThr                              145150155160                                                                  ValLeuAspAlaLeuGlnLeuGlyTyrLysValAsnValIleThrAsp                              165170175                                                                     GlyCysArgGlyValAsnIleGlnProGlnAspSerAlaHisAlaPhe                              180185190                                                                     MetGluMetSerAlaAlaGlyAlaThrLeuTyrThrLeuAlaAspTrp                              195200205                                                                     GluGluThrGlnGly                                                               210                                                                           __________________________________________________________________________

What is claimed is:
 1. An isolated nucleic acid encoding an M.tuberculosis pyrazinamidase (PZase) polypeptide having the amino acidsequence of SEQ. ID NO: 2 or a fragment thereof having biologicalactivity of SEQ. ID No.
 2. 2. The nucleic acid of claim 1, wherein thenucleic acid has the sequence of FIG. 1 (SEQ ID NO: 1), or degeneratevariants thereof, and encodes the amino acid sequence of FIG. 1 (SEQ IDNO: 2).
 3. An isolated nucleic acid encoding a conservative variation ofSEQ. ID NO: 2 and having a biological activity of SEQ. ID NO:
 2. 4. Anisolated nucleic acid encoding an altered M. tuberculosis PZasepolypeptide having the sequence of SEQ. ID NO: 2, except for a -1frameshift at amino acid position
 96. 5. The nucleic acid of claim 4,wherein the nucleic acid comprises a deletion of nucleotide 288 of SEQ.ID NO:
 1. 6. An isolated nucleic acid encoding an altered M.tuberculosis PZase polypeptide having the amino acid sequence of SEQ IDNO: 2 except for a -1 frameshift at amino acid position.
 7. The nucleicacid of claim 6, wherein the nucleic acid comprises a deletion ofnucleotide 162 of SEQ. ID NO:
 1. 8. An isolated nucleic acid encoding analtered M. tuberculosis PZase polypeptide having the amino acid sequenceof SEQ.ID NO: 2, except for a histidine residue at amino acid position.9. The nucleic acid of claim 8, wherein the nucleic acid comprises achange of G to C at nucleotide 187 of SEQ. ID NO:
 1. 10. An isolatednucleic acid encoding an altered M. tuberculosis PZase polypeptide,wherein the nucleic acid encodes a serine residue at amino acid position138 of SEQ. ID NO:
 2. 11. The nucleic acid of claim 10, wherein thenucleic acid comprises a change of T to A at nucleotide 412 of SEQ. IDNO:
 1. 12. An isolated nucleic acid encoding an altered M. tuberculosisPZase polypeptide, wherein the nucleic acid encodes a polypeptide havingthe sequence of SEQ. ID. NO: 2, except for a proline residue at aminoacid position
 141. 13. The nucleic acid of claim 12, wherein the nucleicacid comprises a change of A to C at nucleotide 422 of SEQ. ID NO: 1.